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Abstract:

Methods and sets of non-biological reagents (elution reagents, tag
isomers, tag reactive groups, crosslinkers) for single or multiplexed
capture and gentle elution of biomolecules. Examples are provided using
amine- and cysteine-reactive reagents for enrichment of proteins,
peptides, and rare peptide modifications.

Claims:

1. A method for labeling and selective enrichment of a biomolecule in a
sample, the method comprising (a) covalently tagging a biomolecule in the
sample with at least one chemical affinity tag selected from a dimethyl
piperidine-(DMP-) based chemical affinity tag or a dimethyl
piperazine-(DMPZ-) based chemical affinity tag, and salts thereof, under
conditions to result in a chemically tagged biomolecule (b) selectively
capturing the chemically tagged biomolecule with either (i) a solid
support to which a chemical affinity tag epitope or analog
thereof-selective antibody is attached, or (ii) a solution comprising a
chemical affinity tag epitope or analog thereof-selective antibody, and
thereafter contacting the solution with a solid support capable of
capturing the chemical affinity tag epitope or analog thereof-selective
antibody, to selectively capture the chemically tagged biomolecule, and
(c) eluting the chemically tagged biomolecule by adding at least one
elution reagent comprising at least one displacement molecule to the
solid support to competitively elute under native conditions an intact
chemically affinity tagged biomolecule.

2. The method of claim 1 where the at least one displacement molecule in
step (c) is a substructure of the chemical affinity tag epitope or analog
thereof.

3. The method of claim 1 where step (a) is performed on a single sample
or is performed on at least two separate samples and the separate samples
are combined prior to step (b) resulting in a multiplex method.

4. The method of claim 1 where the DMP-based chemical affinity tag is
selected from the group consisting of ##STR00005## ##STR00006##
##STR00007## and structural analogs thereof, and salts thereof, where
the DMP-based chemical affinity tag is optionally isotopically labeled.

5. The method of claim 1 where the chemical affinity tag further
comprises a linking group and a reactive group(s), where the chemical
affinity tag labels the biomolecule by at least one of amine, carboxyl,
thiol, carbonyl (aldehyde/ketone), azide, alkyne, cyclic alkyne, and/or
phosphine reactive chemistries.

6. The method of claim 1 further comprising, after step (c), (d) removing
the at least one elution reagent by vacuum drying, desalting with
dialysis, reversed phase chromatography, or size exclusion
chromatography.

7. The method of claim 1 where the biomolecule is at least one of cells,
proteins, peptides, glycans, steroids, nucleotides, sugars, toxins,
lipids, and/or small metabolites.

10. The method of claim 1 where the epitope in step (b) comprises a
fragment, substructure, structural analog, or a derivative of the
chemical affinity tag.

11. The method of claim 1 where the chemical affinity tag added in step
(a) comprises an optional crosslinker.

12. The method of claim 1 further comprising after step (c) or claim 6
further comprising after step (d), performing mass spectroscopy analysis
on the eluted biomolecule.

13. The method of claim 1 wherein the solid support in step (b) is either
a particle that is optionally magnetic, or a surface that is at least one
of plastic, glass, ceramics, silicone, metal, cellulose, or gel.

14. The method of claim 1 further comprising adding at least a second
chemical affinity tag to the sample, where the at least second chemical
affinity tag is different from the at least one chemical affinity tag.

15. The method of claim 1 where the chemical affinity tag is either a
DMP-based chemical affinity tag, having the structure ##STR00008## or a
DMPZ-based chemical affinity tag, having the structure ##STR00009##
where Y, if present, is a straight chain or branched C1-C6
alkyl group or a straight chain or branched C1-C6 alkyl ether
group wherein the carbon atoms of the alkyl group or alkyl ether group
each independently comprise hydrogen, deuterium or fluorine atoms; where
R is any length linker comprised of C, N, O, H between the N-substituted
ring and the reactive group(s); and where each Z is independently
hydrogen, fluorine, chlorine, bromine, iodine, an amino acid side chain,
a straight chain or branched C1-C6 alkyl group that may
optionally contain a substituted or unsubstituted aryl group wherein the
carbon atoms of the alkyl and aryl groups each independently comprise
hydrogen or fluorine atoms, a straight chain or branched C1-C6
alkyl ether group that may optionally contain a substituted or
unsubstituted aryl group wherein the carbon atoms of the alkyl and aryl
groups each independently comprise hydrogen or fluorine atoms or a
straight chain or branched C1-C6 alkoxy group that may
optionally contain a substituted or unsubstituted aryl group wherein the
carbon atoms of the alkyl and aryl groups each independently comprise
hydrogen or fluorine atoms.

16. The method of claim 1 using the displacement molecule in step (c)
having sufficient solubility to permit competitive elution with a
concentration 10 to 1,000,000,000 times the affinity binding constant of
the chemical affinity tag epitope or analog thereof selective antibody
affinity to the chemical affinity tag.

17. The method of claim 1 where step (c) is performed at a pH of 4 to 10.

18. The method of claim 1 where each of the dimethyl piperidine-(DMP-)
based chemical affinity tag and dimethyl piperazine-(DMPZ-) based
chemical affinity tag is a free base or any salt thereof.

19. The method of claim 1 using an elution reagent containing at least
one buffer.

21. The method of claim 1 wherein the at least one chemical affinity tag
of step (a) is iodoTMT; the biomolecule in the sample is at least one
S-nitrosylated peptide with S-nitrosyl groups reduced to generate free
sulfhydryl groups prior to step (a), the free sulfhydryl groups reactive
with iodoTMT to chemically tag the biomolecule; the chemically tagged
biomolecule is selectively captured using an anti-TMT antibody resin in
step (b); and the chemically tagged biomolecule is eluted in step (c).

22. The method of claim 21 wherein the sample is a plurality of blood
serum samples; the iodoTMT comprises a set of isobaric chemical affinity
tags used to label each of the samples; after covalently tagging the
biomolecule in each of the samples in step (a), the samples are combined
prior to step (b); and following step (c) of claim 1 or step (d) of claim
7, performing mass spectroscopy analysis on the eluted biomolecule for a
relative quantitation of peptides in a single MS analysis.

23. The method of claim 22, wherein the sample is subjected to abundant
serum protein depletion prior to covalently tagging the biomolecule in
each of the samples.

24. A kit comprising (a) a chemical affinity tag, the chemical affinity
tag selected from a dimethyl piperidine-(DMP-) based chemical affinity
tag or a dimethyl piperazine-(DMPZ-) based chemical affinity tag, (b) a
chemical affinity tag epitope or analog thereof-selective antibody
optionally on a solid support, (c) at least one displacement molecule
selected from the group consisting of a substructure of the chemical
affinity tag epitope and epitope analogs selected from the group
consisting of piperidine, cis-2,6-dimethyl piperidine, 2-S-methyl
piperidine, 2-methyl piperidine, 2,2,4,4-tetramethyl piperidine,
triethylamine (TEA), N,N-disopropylethylamine (DIPEA), triethylammonium
bicarbonate (TEAB), triethylammonium acetate (TEAA), and combinations
thereof, and (d) instructions for use of the kit to selectively label and
enrich biomolecules in a sample.

25. The kit of claim 24 where the affinity tag in (a) is
isotopically-labeled.

26. The kit of claim 24 where the dimethyl piperidine- (DMP-) or dimethyl
piperazine- (DMPZ-) based chemical affinity tag is bound or linked to at
least one biological entity or a reagent for modification of a biological
entity.

27. The kit of claim 24 where the at least one displacement molecule has
sufficient solubility to permit competitive elution with a concentration
10 to 1,000,000,000 times the affinity binding constant of the chemical
affinity tag epitope or analog thereof-selective antibody affinity to the
chemical affinity tag.

[0003] The method used multiplexed antibody-based capture of a series of
isomers or isotopically labeled variants of dimethyl piperidine- or
dimethyl piperazine, collectively DMP-, based chemical affinity tags. The
DMP tags are detection and capture bioconjugation reagents; they contain
a small, non-biological epitope at one end of the tag, and a reactive
group at the other end of the tag. The DMP tags are strong antigens for
antibodies that are developed against the epitope on the affinity tag.
The antibodies are immobilized, and labeled samples are captured with the
immobilized antibody. The labeled samples are then washed and
competitively eluted under native conditions, e.g. in the absence of a
detergent such as sodium dodecyl sulfate or a denaturant such as urea,
i.e. gentle elution, with a small molecule version of the epitope which
is comprised of the tag itself or a fragment, substructure, or structural
analog of the epitope. The elution reagent is removed by methods known in
the art, e.g., vacuum drying, desalting with dialysis or reversed phase
or size exclusion chromatography. Multiple versions of the chemical tags
are constructed with heavy stable isotopes and/or unique linkers between
the epitope and reactive groups, allowing labeling of multiple samples,
mixing of these samples, and multiplexed capture prior to mass
spectrometry analysis. The reactive groups specifically label amine,
carboxyls, carbonyls, azides, phosphines, alkynes, or cyclic alkynes. The
competitive elution reagent may be removed by dialysis, size-exclusion
desalting resin, precipitation, or vacuum drying.

[0004] The method selectively captures one or more structural analogs
(e.g. β-Ala) or isotopically-labeled isomers of a DMP tag in a
single or multiplexed capture reaction from a biological matrix using an
antibody that is epitope-selective, i.e., dimethyl piperidine-selective
and analogs thereof.

[0005] Biomolecules, including protein, peptide, nucleic acid,
oligosaccharide, glycoprotein, lipid, carbohydrate, hormones, toxins, and
cells are studied by multiple analytical methods to determine their
structure and function, to characterize any modifications and the effects
of these modifications (e.g. structure-function relationships), and to
quantify changes in the biomolecule levels and their interactions in
response to growth, development, disease, treatment, and other
environmental factors. These biomolecules may be present in the
environment, in blood or serum, in tissue, in cells, in subcellular
compartments, and/or in cellular complexes.

[0006] Many reagents and research tools have been developed to enrich
biomolecules of interest for these studies. These reagents include
biotin, desthiobiotin, nitrophenyl reagents, and other bioconjugation
derivatives. In one example, a biomolecule is conjugated with a biotin
reagent ("bait"), added to a complex sample, and then captured through
the substantially irreversible 10-14 mol/L binding interaction with
streptavidin-coated beads or surfaces to co-enrich other molecules
("prey") that may bind or interact with the bait molecule. In another
example, an antibody is conjugated with a biotinylating reagent so that
it may be captured with an immobilized biotin-binding protein. The
antibody is then added to a biological sample, wherein a target antigen
is then captured from the complex protein sample with streptavidin-coated
beads or surfaces for characterization or quantification.

[0007] Prior to elution of bound material, particles may be washed with
non-denaturing detergents, high or low salt, pH, or solvents to reduce
nonspecific background, and then the antigen can be eluted with strong
acidic or basic pH conditions or denaturing concentrations of detergent.
In one example, a biotinylated protein or peptide can be enriched with
streptavidin coated beads, but can only be recovered through heating at
90° C. in combination with acidic buffers, organic solvents,
strong detergents, and excess competitive biotin. Desthiobiotin,
iminobiotin, monomeric avidin, and nitrosylated streptavidin are all
reagents with lower affinity interactions, but these still require heat,
extreme pH, and/or other harsh elution conditions to recover biotinylated
proteins or peptides efficiently. This strategy will also capture
endogenously biotinylated molecules, which may interfere with the
analysis.

[0008] Alternatively, fusion proteins are expressed with N- or C-terminal
affinity tags, such as 6×His, FLAG, glutathione S-transferase
(GST), or hemagglutinin (HA). Each of these tags can be captured with
affinity resins to purify expressed fusion proteins. In one example, a
protein with a 6×His tag can be captured through the strong
interaction between the imidazole ring on histidine residues in the
affinity tag and a nickel or cobalt chelated immobilized metal affinity
column (IMAC). This interaction is not affected by denaturing conditions,
allowing aggregates of His-tagged protein to be solubilized in 8M urea
denaturing conditions, captured with Ni- or Co-IMAC, refolded on column
by reducing the urea concentration, and then competitively eluted with
imidazole. In another example, a fusion protein expressed with GST, FLAG,
or HA affinity tag can be purified with glutathione resin or immobilized
anti-FLAG or anti-HA antibody resins, respectively, and then
competitively eluted with free glutathione, FLAG peptide, or HA peptide.
When the antigen of interest is present at a very low concentration in a
complex sample, competitive and selective elution conditions may be
necessary to reduce the background of non-specific biomolecules that may
co-elute under harsh or denaturing conditions. Competitive elution
conditions improve the quantitative recovery of the analyte and the
specificity of elution of biomolecules.

[0009] The analysis of rare protein modifications and of protein-protein
interactions is complicated by the low stoichiometry and transient nature
of the interactions. In addition, while many modifications and
interactions may be detected by antibody-based methods, the exact
location and molecular characterization of these modifications or
interactions are difficult to study. In one example, a protein may be
nitrosylated on cysteine residues in response to oxidative stress or
other environmental stimuli. While it is possible to use chemical methods
to specifically biotinylate nitrosylated sites using a "biotin-switch"
methodology, and antibody-based methods to capture a protein of interest
and detect the presence of nitrosylation, it is very difficult to
identify the site of modification and quantitatively monitor the
site-specific changes under different treatment conditions or over time.

[0010] Similarly, a protein-protein interaction can be inferred by the
co-immunocapture of a prey protein with a bait protein, but the molecular
details of this interaction are missing without a chemical means of
tagging the precise sites of interaction. In one example, a
homobifunctional, amine-reactive chemical crosslinker is added to a cell
lysate, a protein of interest is captured with an antibody to that
protein, and other proteins that were co-enriched are assessed by
resolving the enriched sample electrophoretically on a denaturing
polyacrylamide gel, transferring the lane of separated proteins to a
membrane, and then probing this membrane with an antibody against
candidate proteins by Western blot to detect interaction and
co-enrichment. In another example, the crosslinker may contain an
affinity handle so that the sample may be digested and the cross-linked
fragments may be enriched with the affinity handle to allow
identification of the cross-linked fragments and their sites of linkage.
Reagents that allow enrichment and quantitative analysis of these
modifications and interactions may provide critical understanding of
protein structure and interactions.

[0011] The efficiency of capture depends upon the affinity of the binding
interaction, the presence of interfering molecules, and the accessibility
of the binding components. Accessibility may be affected by protein
folding, aggregation, complexes, and incomplete solubilization. In one
example, an antibody is used to capture a protein antigen from a cell
lysate. The efficiency of capture is determined in a complementary assay,
such as ELISA or Western blot, by comparing samples of the starting
lysate, the captured protein sample, and the depleted lysate. Because
capture or depletion is often incomplete, reagent controls may be used to
account for differences in capture efficiency between conditions. In one
example, a version of the targeted protein is expressed or tagged with a
unique dye, isotopic signature, or other modification and then spiked
into all samples, captured along with the native target of interest, and
then quantified to assess and normalize capture efficiency across all
samples. In a similar example, multiple samples from multiple conditions
are modified with a set of similar but distinct tags, the uniquely tagged
samples are combined, the antigens are captured simultaneously in one
reaction, and then the relative intensities of the unique tags are
quantified after elution to compare protein amounts. This multiplexed
capture strategy allows many samples to be enriched and analyzed
simultaneously from one capture experiment. This strategy reduces the
variability introduced with separate capture and elution experiments.

[0012] It is often desirable to elute a captured biomolecule or captured
cell in its native form in order to preserve structural features,
enzymatic function, or cellular viability. In one example, a biomolecule
may be labeled with a modified biotin reagent, such as desthiobiotin,
and/or captured with a modified biotin binding protein, such as monomeric
avidin, which have a lower binding affinity than the native avidin-biotin
interaction. After capture, the bound biomolecule may be competitively
eluted with free biotin. Despite the lower affinity, this binding
interaction is cooperative and high affinity, making it difficult to
dissociate with high recovery without heat or extreme pH conditions. An
elution reagent that can quickly and efficiently elute captured molecules
under gentle conditions may better preserve the target analyte structure
and function.

[0013] Similarly, after capture and elution of a molecule, it is often
desirable to remove or neutralize the elution reagent without affecting
sample quality or recovery. In one example, a desthiobiotin-labeled
sample may be eluted from streptavidin with a solution of sodium dodecyl
sulfate (SDS), a strong denaturing detergent. In some analyses, such as
Western blotting, this detergent may be advantageous for solubilization
and denaturation. However, in other analyses, such as mass spectrometric
analysis, even low quantities of this detergent can prevent detection of
the analyte and cause deterioration of the instrument performance. For
standard liquid chromatography-mass spectrometric (LC-MS) analysis, it is
often necessary to neutralize and remove the elution reagent in order to
avoid interferences and the need to replace chromatography consumables
and/or repair instrumentation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The patent or application file contains at least one drawing
executed in color. A Petition under 37 C.F.R. §1.84 requesting
acceptance of the color drawing is being filed separately. Copies of this
patent or patent application publication with color drawing(s) will be
provided by the Office upon request and payment of the necessary fee.

[0037] One embodiment is a method for labeling and selective enrichment of
a biomolecule in a sample. The method (a) covalently tags a biomolecule
in the sample with at least one dimethyl piperidine- or dimethyl
piperazine- (DMP-) based chemical affinity tag under conditions to result
in a DMP-based chemically tagged biomolecule, (b) then selectively
captures the DMP-based chemically tagged biomolecule with either (i) a
solid support to which a DMP epitope or analog thereof-selective antibody
is attached, or (ii) a solution of a DMP epitope or analog
thereof-selective antibody, and then contacting the solution with a solid
support capable of capturing the DMP epitope or analog thereof-selective
antibody to selectively capture the DMP-based chemically tagged
biomolecule, and (c) eluting the DMP-based chemically tagged biomolecule
by adding at least one elution reagent comprising a displacement molecule
to the solid support to competitively selectively elute under native
conditions an intact DMP-based chemically affinity tagged biomolecule.

[0038] In one embodiment, the method reversibly captures a biomolecule
labeled with an N-substituted piperidine compound or a salt thereof. The
N-substituted piperidine has the following structure,

##STR00001##

where R is any length linker comprised of C, N, O, H between the
N-substituted ring and the reactive group(s); and where each Z is
independently hydrogen, fluorine, chlorine, bromine, iodine, an amino
acid side chain, a straight chain or branched C1-C6 alkyl group
that may optionally contain a substituted or unsubstituted aryl group
where the carbon atoms of the alkyl and aryl groups each independently
comprise hydrogen or fluorine atoms; a straight chain or branched
C1-C6 alkyl ether group that may optionally contain a
substituted or unsubstituted aryl group where the carbon atoms of the
alkyl and aryl groups each independently comprise hydrogen or fluorine
atoms; or a straight chain or branched C1-C6 alkoxy group that
may optionally contain a substituted or unsubstituted aryl group wherein
the carbon atoms of the alkyl and aryl groups each independently comprise
hydrogen or fluorine atoms. The method (a) covalently tags a biomolecule
in the sample with at least one N-substituted piperidine compound or a
salt thereof-based chemical affinity tag under conditions to result in a
N-substituted piperidine compound or a salt thereof-based chemically
tagged biomolecule, (b) selectively captures the N-substituted piperidine
or a salt thereof based chemically tagged biomolecule with either (i) a
solid support to which an N-substituted piperidine compound or a salt
thereof epitope or analog thereof-selective antibody is attached, or (ii)
a solution comprising an N-substituted piperidine compound or a salt
thereof epitope or analog thereof-selective antibody, and then contacting
the solution with a solid support capable of capturing the N-substituted
piperidine compound or a salt thereof epitope or analog thereof-selective
antibody to selectively capture the N-substituted piperidine compound or
a salt thereof-based chemically tagged biomolecule, and (c) elutes the
N-substituted piperidine compound or a salt thereof-based chemically
tagged biomolecule by adding at least one elution reagent comprising at
least one displacement molecule to the solid support to competitively
selectively elute under native conditions an intact N-substituted
piperidine compound or a salt thereof-based chemically affinity tagged
biomolecule.

[0039] In one embodiment, the N-substituted piperidine compound or a salt
thereof-based chemically affinity tagged biomolecule is a DMP-based
chemical affinity tag.

[0040] In one embodiment, at least one displacement molecule in step (c)
is a substructure of the DMP epitope or analog thereof. In embodiments,
step (a) is performed on a single sample, or step (a) is performed on at
least two separate samples and the separate samples are combined prior to
step (b) resulting in a multiplex method.

[0041] In one embodiment, the DMP-based chemical affinity tag is among the
compounds of FIG. 7 and structural analogs thereof and among the
compounds of FIG. 8 and structural analogs thereof, where the DMP-based
chemical affinity tag is optionally isotopically labeled. In one
embodiment, the DMP-based affinity tag has a linking group and a reactive
group, where the DMP-based chemical affinity tag labels the biomolecule
by at least one of amine, carboxyl, thiol, carbonyl (aldehyde/ketone),
azide, alkyne, cyclic alkyne, and/or phosphine reactive chemistries.

[0042] In one embodiment, the elution of step (c) occurs at pH ranging
from about 4 to about 10. In contrast, biotin-containing capture reagents
such as ICAT require elution at pH less than 4

[0043] In one embodiment, the method further comprises, after step (c), a
step (d) where the elution reagent(s) are removed by vacuum drying or
desalting with dialysis or reversed phase or size exclusion
chromatography. The elution reagents are volatile, and thus are readily
removed by, e.g., vacuum drying.

[0045] In one embodiment, the elution reagent contains at least one
displacement molecule. In one embodiment, the displacement molecule(s) is
a substructure of the DMP epitope and epitope analogs. In one embodiment,
the displacement molecule is piperidine, 2-S-methyl piperidine, 2-methyl
piperidine, c is 2,6-dimethyl piperidine, 2,2,4,4-tetramethyl piperidine,
triethylamine, and/or diisopropylethylamine. In one embodiment, the
displacement molecule is not a substructure of the DMP epitope and
epitope analogs. In one embodiment, the displacement molecule is
triethylamine (TEA), N,N-disopropylethylamine (DIPEA), triethylammonium
acetate (TEAA), and/or triethylammonium bicarbonate (TEAB). In one
embodiment, the elution reagent comprises more than one displacement
molecule, where the displacement molecules may be a combination of a
substructure of the DMP epitope and epitope analogs, a combination of
compounds that are not a substructure of the DMP epitope and epitope
analogs, and combinations of substructure of the DMP epitope and epitope
analogs and compounds that are not a substructure of the DMP epitope and
epitope analogs. In one embodiment, the elution reagent contains at least
one buffer, e.g., ((hydroxymethyl)aminomethane) (Tris),
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),
2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid
(TES), phosphate, 2-(N-morpholino)ethanesulfonic acid (MES),
3-morpholinopropane-1-sulfonic acid (MOPS),
1,4-piperazinediethanesulfonic acid (PIPES), bicarbonate, carbonate,
N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine (tricine),
N,N-(bis(2-hydroxyethyl)glycine (bicine), triethylammonium acetate
(TEAA), and/or triethylammonium bicarbonate (TEAB), etc.

[0046] In one embodiment, the DMP epitope-selective antibody in step (b)
is a glycoform, Fab fragment, or derivative thereof. In addition, the DMP
epitope to which the described antibody reacts and is present in step (b)
of the method can be a fragment, substructure, structural analog, or a
derivative of the DMP-based affinity tag.

[0048] In one embodiment, the method further comprises after the elution
of step (c) or after the removal of elution reagent in step (d),
performing mass spectroscopy analysis on the eluted biomolecule.

[0050] In one embodiment, the method may further add at least a second
DMP-based chemical affinity tag to the sample, different from the first
DMP-based affinity tag.

[0051] In one embodiment of the method, the DMP-based chemical affinity
tag has the piperidine structure

##STR00002##

where R is any length linker comprised of C, N, O, H between the
N-substituted ring and the reactive group(s); and where each Z is
independently hydrogen, fluorine, chlorine, bromine, iodine, an amino
acid side chain, a straight chain or branched C1-C6 alkyl group
that may optionally contain a substituted or unsubstituted aryl group
wherein the carbon atoms of the alkyl and aryl groups each independently
comprise hydrogen or fluorine atoms, a straight chain or branched
C1-C6 alkyl ether group that may optionally contain a
substituted or unsubstituted aryl group wherein the carbon atoms of the
alkyl and aryl groups each independently comprise hydrogen or fluorine
atoms, or a straight chain or branched C1-C6 alkoxy group that
may optionally contain a substituted or unsubstituted aryl group where
the carbon atoms of the alkyl and aryl groups each independently comprise
hydrogen or fluorine atoms.

[0052] In one embodiment, the displacement molecule in step (c) is
sufficiently soluble to permit competitive elution with a concentration
10 to 1,000,000,000 times the affinity binding constant of the DMP
epitope or analog thereof selective antibody affinity to the DMP-based
chemical affinity tag. In one embodiment, step (c) is performed at a pH
in the range of about 4 to about 10.

[0053] In one embodiment, the method (a) covalently tags a biomolecule in
the sample with at least one dimethyl piperazine- (DMPZ-) based chemical
affinity tag under conditions to result in a DMPZ-based chemically tagged
biomolecule, (b) selectively captures the DMPZ-based chemically tagged
biomolecule with either (i) a solid support to which a DMPZ epitope or
analog thereof-selective antibody is attached, or (ii) a solution
comprising a DMPZ epitope or analog thereof-selective antibody, and
thereafter contacting the solution with a solid support capable of
capturing the DMPZ epitope or analog thereof-selective antibody to
selectively capture the DMPZ-based chemically tagged biomolecule, and (c)
elutes the DMPZ-based chemically tagged biomolecule by adding at least
one elution reagent comprising at least one displacement molecule to the
solid support to competitively selectively elute under native conditions
an intact DMPZ-based chemically tagged biomolecule.

[0054] In one embodiment, the method reversibly captures a biomolecule
labeled with an N-substituted piperazine compound or a salt thereof
having the following structure

##STR00003##

where Y is a straight chain or branched C1-C6 alkyl group or a
straight chain or branched C1-C6 alkyl ether group, where the
carbon atoms of the alkyl group or alkyl ether group each independently
comprise hydrogen, deuterium or fluorine atoms; where R is any length
linker comprised of C, N, O, H between the N-substituted ring and the
reactive group(s); and where each Z is independently hydrogen, fluorine,
chlorine, bromine, iodine, an amino acid side chain, a straight chain or
branched C1-C6 alkyl group that may optionally contain a
substituted or unsubstituted aryl group, where the carbon atoms of the
alkyl and aryl groups each independently comprise hydrogen or fluorine
atoms; a straight chain or branched C1-C6 alkyl ether group
that may optionally contain a substituted or unsubstituted aryl group
where the carbon atoms of the alkyl and aryl groups each independently
comprise hydrogen or fluorine atoms; or a straight chain or branched
C1-C6 alkoxy group that may optionally contain a substituted or
unsubstituted aryl group where the carbon atoms of the alkyl and aryl
groups each independently comprise hydrogen or fluorine atoms; where the
method (a) covalently tags a biomolecule in the sample with at least one
N-substituted piperazine compound or a salt thereof-based chemical
affinity tag under conditions to result in a N-substituted piperazine
compound or a salt thereof-based chemically tagged biomolecule, (b)
selectively captures the N-substituted piperazine compound or a salt
thereof-based chemically tagged biomolecule with either (i) a solid
support to which an N-substituted piperazine compound epitope or analog
thereof-selective antibody is attached, or (ii) a solution comprising an
N-substituted piperazine compound epitope or analog thereof-selective
antibody, and thereafter contacting the solution with a solid support
capable of capturing the N-substituted piperazine compound epitope or
analog thereof-selective antibody to selectively capture the
N-substituted piperazine compound or a salt thereof-based chemically
tagged biomolecule, and (c) elutes the N-substituted piperazine compound
or a salt thereof-based chemically tagged biomolecule by adding at least
one elution reagent comprising at least one displacement molecule to the
solid support to competitively selectively elute under native conditions
an intact N-substituted piperazine compound or a salt thereof-based
chemically tagged biomolecule.

[0055] In one embodiment, the N-substituted piperazine compound or a salt
thereof-based chemically affinity tagged biomolecule is a DMPZ-based
chemical affinity tag.

[0056] In one embodiment, the displacement molecule in step (c) is a
substructure of the DMPZ epitope or analog thereof. In one embodiment,
the N-substituted piperazine is a bis-trifluoroacetic acid (TFA) salt, a
bis-HCl salt, a bis-acetic acid salt, a bis-formic acid salt, and/or a
mix of these or other acidic salts.

[0057] In embodiments, step (a) is performed on a single sample, or step
(a) is performed on at least two separate samples and the separate
samples are combined prior to step (b), resulting in a multiplex method.

[0058] In one embodiment, the DMPZ-based chemical affinity tag is
optionally isotopically labeled. In one embodiment, the DMPZ-based
affinity tag has a linking group and a reactive group, where the
DMPZ-based chemical affinity tag labels the biomolecule by amine,
carboxyl, thiol, carbonyl (aldehyde/ketone), azide, alkyne, cyclic
alkyne, and/or phosphine reactive chemistries.

[0059] In one embodiment, the elution of step (c) occurs at pH ranging
from about 4 to about 10.

[0060] In one embodiment, the method further comprises, after step (c), a
step (d) where the elution reagent(s) are removed by vacuum drying or
desalting with dialysis or reversed phase or size exclusion
chromatography. In embodiments, the elution reagents are volatile, and
thus are readily removed by, e.g., vacuum drying.

[0061] In embodiments, the biomolecule that is tagged is a cell, protein,
peptide, glycan, steroid, nucleotide, sugar, toxin, lipid, and/or small
metabolite.

[0062] In one embodiment, the elution reagent contains at least one
displacement molecule. In one embodiment, the displacement molecule(s) is
a substructure of the DMPZ epitope and epitope analogs.

[0063] In one embodiment, the displacement molecule is piperidine,
2-S-methyl piperidine, 2-methyl piperidine, cis-2,6-dimethyl piperidine,
2,2,4,4-tetramethyl piperidine, triethylamine (TEA), and/or
diisopropylethylamine. In one embodiment, the displacement molecule is
not a substructure of the DMPZ epitope and epitope analogs. In one
embodiment, the displacement molecule is triethylamine (TEA),
N,N-disopropylethylamine (DIPEA), triethylammonium acetate (TEAA), and/or
triethylammonium bicarbonate (TEAB). In one embodiment, the elution
reagent comprises more than one displacement molecule, where the
displacement molecules may be a combination of a substructure of the DMPZ
epitope and epitope analogs, a combination of compounds which are not a
substructure of the DMPZ epitope and epitope analogs, and combinations of
substructure of the DMPZ epitope and epitope analogs and compounds which
are not a substructure of the DMPZ epitope and epitope analogs. In one
embodiment, the elution reagent contains at least one buffer, e.g., Tris,
HEPES, TES, phosphate, MES, MOPS, PIPES, bicarbonate, carbonate, tricine,
bicine, TEAB, TEAA, etc.

[0064] In one embodiment, the DMPZ epitope-selective antibody in step (b)
is a glycoform, Fab fragment or derivatives thereof. In addition, the
DMPZ epitope to which the described antibody reacts and is present in
step (b) of the method comprises a fragment, substructure, structural
analog, or a derivative of the DMPZ-based affinity tag.

[0065] In embodiments, the DMPZ-based chemical affinity tag added in step
(a) has an optional crosslinker.

[0066] In one embodiment, the method further comprises after the elution
of step (c) or after the removal of elution reagent in step (d),
performing mass spectroscopy analysis on the eluted biomolecule.

[0067] In embodiments, the solid support of step (b) is a particle, e.g.,
a magnetic particle, or a plastic, glass, ceramics, silicone, metal,
and/or cellulose surface.

[0068] In one embodiment, the method may further add at least a second
DMPZ-based chemical affinity tag to the sample, different from the first
DMPZ-based affinity tag.

[0069] In one embodiment, the method further adds at least a second
N-substituted piperazine compound or a salt thereof-based chemical
affinity tag to the sample, where the second tag is different from the
first tag.

[0070] In one embodiment of the method, the DMPZ-based chemical affinity
tag has the piperazine structure

##STR00004##

where Y is a straight chain or branched C1-C6 alkyl group or a
straight chain or branched C1-C6 alkyl ether group, where the
carbon atoms of the alkyl group or alkyl ether group each independently
comprise hydrogen, deuterium or fluorine atoms; where R is any length
linker comprised of C, N, O, H between the N-substituted ring and the
reactive group(s); and where each Z is independently hydrogen, fluorine,
chlorine, bromine, iodine, an amino acid side chain, a straight chain or
branched C1-C6 alkyl group that may optionally contain a
substituted or unsubstituted aryl group wherein the carbon atoms of the
alkyl and aryl groups each independently comprise hydrogen or fluorine
atoms, a straight chain or branched C1-C6 alkyl ether group
that may optionally contain a substituted or unsubstituted aryl group
wherein the carbon atoms of the alkyl and aryl groups each independently
comprise hydrogen or fluorine atoms, or a straight chain or branched
C1-C6 alkoxy group that may optionally contain a substituted or
unsubstituted aryl group where the carbon atoms of the alkyl and aryl
groups each independently comprise hydrogen or fluorine atoms.

[0071] In one embodiment, the displacement molecule in step (c) is
sufficiently soluble to permit competitive elution with a concentration
10 to 1,000,000,000 times the affinity binding constant of the DMPZ
epitope or analog thereof selective antibody affinity to the DMPZ-based
chemical affinity tag. In one embodiment, step (c) is performed at a pH
in the range of about 4 to about 10.

[0072] In one embodiment, a kit comprises (a) a dimethyl piperidine-
(DMP-) based chemical affinity tag, (b) a DMP epitope-selective antibody
optionally on a solid support, and (c) a substructure of the DMP epitope
and epitope analogs selected from the group consisting of piperidine,
2-S-methyl piperidine, 2-methyl piperidine, 2,2,4,4-tetramethyl
piperidine, triethylamine, diisopropylethylamine, and combinations
thereof, and (d) instructions for using the kit to selectively label and
enrich biomolecules in a sample.

[0073] In one embodiment, the kit comprises (a) a dimethyl piperazine-
(DMPZ-) chemical affinity tag, (b) a DMPZ epitope-selective antibody
optionally on a solid support, and (c) a substructure of a DMPZ epitope
or analog thereof, and (d) instructions for using the kit to selectively
label and enrich biomolecules in a sample.

[0074] In one embodiment, the kit comprises (a) an affinity tag, (b) an
anti-affinity tag antibody optionally on a solid support, and (c) a
substructure of a dimethyl piperazine or analog thereof, or dimethyl
piperidine or analog thereof, and (d) instructions for using the kit to
selectively label and enrich biomolecules in a sample.

[0075] In embodiments, the affinity tag in the kit is
isotopically-labeled. In one embodiment of the kit, the dimethyl
piperidine- (DMP-) or dimethyl piperazine- (DMPZ-) based chemical
affinity tag is bound or linked to at least one biological entity or a
reagent for modification of a biological entity. In one embodiment of the
kit, the substructure of the DMP epitope or analogs thereof, or the
substructure of a DMPZ epitope or analog thereof, has sufficient
solubility to permit competitive elution with a concentration 10 to
1,000,000,000 times the affinity binding constant of the DMP epitope or
analog thereof selective antibody, or DMPZ epitope or analog thereof
selective antibody affinity to the DMP-based chemical affinity tag or
DMPZ-based chemical affinity tag.

[0076] In FIG. 1A, antibody is incubated with a sample containing the
biomolecule labeled with an epitope tag. A Protein A/G-coated resin is
then added to the sample to capture the antibody with its bound antigen.
Elution is performed by addition of the small molecule elution reagent
and centrifugation to separate the resin from the released analyte in
solution. In FIG. 1B, the antibody is coupled directly to the resin, and
this resin is incubated with the sample containing the labeled
biomolecule. Elution is performed as described above.

[0079] FIG. 4A shows proteins from an A549 cell lysate that were alkylated
on cysteine residues with an iodoacetyl TMT reagent, digested with
trypsin, and then captured with anti-TMT immobilized antibody resin.
Peptides were washed with Tris buffered saline (TBS), Rapigest detergent
(Waters), or CHAPS detergent and eluted with 0.4% trifluoracetic acid (pH
about 2), or washed with TBS and water prior to elution with DMP at the
indicated concentrations and pH. Eluted peptides were analyzed by
LC-MS/MS on a Thermo Scientific Orbitrap XL and data was interpreted with
Thermo Scientific Proteome Discoverer 1.3 to identify the eluted peptides
and the number of iodoTMT labeled peptides specifically eluted
(percentage shown above each bar). In FIG. 4B, a cell lysate was
S-nitrosylated in vitro with nitroglutathione, the free thiols were
irreversibly alkylated with iodoacetamide, the nitroso groups were
reduced and selectively released with ascorbic acid, the newly exposed
cysteines were irreversibly alkylated with iodoTMT, and the samples were
then reduced to remove disulfide linkages, alkylated with iodoacetamide,
and digested with trypsin. These peptide samples were enriched with the
anti-TMT antibody, and samples of the load, the flowthrough, and the
non-specific and specific elutions were assessed for peptide recovery and
specificity.

[0080] In FIG. 5, showing multiplexed enrichment of S-nitrosylated
peptides, cell lysates from a mammalian cell line were treated with
vehicle or lipopolysaccharide (LPS) to stimulate S-nitrosylation. A
portion of the lysate from untreated cells was also S-nitrosylated in
vitro with nitrocysteine (SNOC) as a positive control. As described
above, free thiols were alkylated with iodoacetamide, the nitroso groups
were selectively released with ascorbic acid, and the newly exposed
cysteines were alkylated with an isobaric set of iodoTMT6 reagents in
duplicate. Specifically, the cells treated with vehicle were alkylated
with iodoTMT6-126 and iodoTMT6-129, the LPS stimulated cells
were alkylated with iodoTMT6-127 and iodoTMT6-129, and the SNOC
treated lysates were alkylated with iodoTMT6-128 and
iodoTMT6-131. These samples were then reduced, alkylated with
iodoacetamide, combined into one tube, and digested with trypsin. These
mixed peptide samples were then enriched with the anti-TMT antibody in
one reaction, and the peptides were identified and quantified by the
126-131 Dalton iodoTMT reporter ions. FIG. 5A shows the localization of
the site of modification of peptide GCITIIGGGDTATC*CAK (SEQ ID NO:1) from
phosphoglycerate kinase 1. The other two cysteines in this peptide were
blocked with iodoacetamide, and the site of iodoTMT labeling indicates
the exact site of S-nitrosylation. FIG. 5B shows quantification of the
peptide from FIG. 5A across three conditions in duplicate. FIG. 5C shows
quantification of peptide C*MMAQYNR (SEQ ID NO:2) from
stress-induced-phosphoprotein 1 (*=site of iodoTMT incorporation).

[0081] FIG. 6 shows chemical structures of piperidine elution reagents.
Multiple variants and structural analogues of piperidine reagents were
evaluated for their ability to elute bound TMT-labeled protein and
peptide. The method is also applicable to using a series of isomers or
isotopically labeled variants of piperazine-based chemical affinity tags.

[0084] FIG. 9 shows a range of chemical reactivities besides the amine and
two cysteine-reactive chemistries described above. All of the reagents
contain dimethyl piperidine for the purpose of example, but could also
contain other N-substituted piperidine or N-substituted piperazine.

[0086] FIG. 11 shows chemical structures for cysteine-reactive tandem mass
tags (cysTMT) labeling in one embodiment. The dithiopyridine chemistry of
these tags selectively reacts with thiols of cysteine residues through a
reversible disulfide linkage. These tags include a non-isotopically
labeled reagent (cysTMTzero) and six cysTMTsixplex reagents with unique
distributions of heavy isotopes so that they weigh the same (i.e. are
isobaric) but yield unique reporter ions. Biological samples labeled with
cysTMTzero and any one of the cysTMTsixplex reagents can be combined and
then selectively captured and analyzed as a duplex pair, with identical
peptides from each sample separated by 5 Da. Alternatively, six samples
can be labeled with the six isobaric reagents, pooled, and then captured
and analyzed as one multiplexed sample.

[0087] FIG. 12 shows an example work flow for mass spectrometry analysis
using cysTMT reagents in one embodiment. These reagents can be used to
label intact proteins from multiple samples before pooling the samples
for processing, multiplexed enrichment, and LC-MS/MS analysis.

[0088] FIG. 13 shows chemical structures for iodoacetyl tandem mass tags
(iodoTMT) labeling in one embodiment. These tags are analogous to the
cysTMT reagents, except that the iodoacetyl chemistry is used to label
thiols irreversibly.

[0089] FIG. 14 shows an example work flow for mass spectrometry analysis
using iodoTMT reagents. The iodoTMT reagents are less sensitive to
quenching by tris(2-carboxyethyl)phosphine (TCEP), so that the TCEP
reducing agent does not need to be removed by dialysis or desalting
before labeling with iodoTMT chemical tags.

[0090] FIGS. 15A-B show iodoTMT reagent labeling efficiency and
specificity. The iodoTMT reagent labeling is sensitive to stoichiometry
and pH conditions. Excess reagent or reaction conditions significantly
higher or lower than pH 8.0 will result in non-selective labeling at
lysines, histidines, aspartates, and glutamate residues.

[0091] FIGS. 16A-B show iodoTMT-labeled peptide enrichment and protein
identification comparison. While only a subset of peptides contain
cysteine residues, the reduction in complexity allowed by selective
labeling and enrichment of cysteine-containing peptides results in a
greater number of proteins identified from a given number of peptides
identified.

[0092] FIG. 17 shows an example of S-nitrosylation Western blotting. A
complex cell lysate was treated in vitro with vehicle (lanes 1,2) or
nitroglutathione (lanes 3,4) and then free thiols were blocked with
iodoacetamide. Nitrosylated thiols can then be labeled with iodoTMT after
the selective reduction of nitrosylated thiols with ascorbate (lanes
2,4).

[0093]FIG. 18 shows the improved selectivity of enrichment of
S-nitrosylated peptides with a soft competitive elution strategy
according to one embodiment, instead of acidic elution buffer.

[0094] FIG. 19 shows the chemical reactivity of hydrazide and alkoxyamine
reagents for the selective labeling, enrichment, identification, and
quantification of peptides, glycans, lipids, steroids, nucleotides, and
other biomolecules containing aldehydes and ketones.

[0098] In one embodiment, the disclosed methods and kits are used to
prepare biomolecules for subsequent mass spectrometry (MS) analysis. In
one embodiment, the biomolecules are prepared using Thermo Scientific's
Tandem Mass Tag reagents (TMT). For example, the Thermo Scientific
Cysteine-Reactive Tandem Mass Tag (cysTMT) reagents enable selective
labeling and relative twoplex to sixplex mass spectrometry (MS)
quantitation of cysteine-containing peptides derived from complex
biological samples. The cysTMT reagents label only free sulfhydryl groups
on cysteine residues, in contrast to the amine-reactive TMT®
reagents, which in other embodiments, may also be used. To selectively
analyze the cysteine-labeled peptides, the sample is enriched using the
Thermo Scientific Immobilized Anti-TMT Antibody Resin. The cysTMT
Reagents and Anti-TMT Resin are effective for reducing sample complexity,
improving dynamic range and studying cysteine modifications. Using this
approach of thiol labeling, affinity enrichment and quantitation is
similar to isotope-coded affinity tags (ICAT).

[0099] Each isobaric cysTMT sixplex reagent within a set has the same
nominal parent mass and is composed of a sulfhydryl-reactive
pyridyldithio group, a MS-neutral spacer arm and an MS/MS reporter (FIG.
11). FIG. 11 shows chemical structure of the Thermo Scientific cysTMT
Label Reagents. Functional regions of the reagent structure with the
isotope positions, MS/MS fragmentation sites and collision-induced
reporter ions for each reagent. The molecular weight of the intact cysTMT
zero reagent is 410.13 Da and the cysTMTsixplex is 416.13 Da. The
reagents label proteins prepared from up to six biological samples or
treatments, which are combined into one sample for the quantitative
analysis of relative protein expression. During the MS/MS stage of
acquisition to derive fragment ions and sequence information, a unique
reporter ion mass also is generated (e.g., 126-131 Da for the cysTMT6
Isobaric Label Reagents). These reporter ions are in the low mass region
of the MS/MS spectrum, providing information on relative protein
expression levels.

[0100] The cysTMTzero and cysTMTsixplex Reagents are used as isotopic
"light" and "heavy" duplex tags for quantitation at the MS stage. These
tags enable quantitation of protein expression changes in cell-based and
tissue samples that might not be amenable to metabolic isotopic labeling
strategies (e.g., SILAC). For example, to label and prepare samples for
analysis (FIG. 12), protein extracts are isolated from cultured cells or
tissues. After proteins are reduced and desalted, each sample is
individually labeled. Excess tag is removed by SDS-PAGE, gel filtration
or the Thermo Scientific Pierce Detergent Removal Spin Columns. For
LC-MS/MS analysis proteins are digested with a site-specific
endoproteinase. After digestion, labeled peptides are enriched using the
Immobilized Anti-TMT Antibody Resin and eluted using the disclosed
method. Data acquisition is performed on a Thermo Scientific LTQ-Orbitrap
or Velos-Orbitrap Mass Spectrometer, and data analysis software is used
for protein identification and relative quantitation of the six samples
via reporter ions.

EXAMPLE 1

[0101] The DMP tags are non-biological detection and capture
bioconjugation reagents that serve as strong antigens for antibody
development. Multiple hybridomas were developed and identified from mice
injected with BSA labeled with an amine-DMP tag (FIG. 2A). These
antibodies have binding affinities in the pM to nM range, and can be used
for Western blot detection of labeled proteins with little or no
interfering background signal (FIG. 2B, D).

EXAMPLE 2

[0102] An anti-TMT antibody binds tightly to a TMT-labeled surface, but
washing in pM to mM concentrations of DMP and DMP analogs competes for
binding and elutes bound antibody within minutes at room temperature
(FIG. 3A). Anti-TMT antibody resin specifically binds intact protein
labeled with amine-reactive TMT but not unlabeled protein, and the bound
protein can be efficiently eluted with micromolar to millimolar dimethyl
piperidine. As the protein is labeled at nearly all amine sites, more
sites on the protein are likely bound to the resin and more competitor
reagent is necessary to elute the bound protein (FIG. 3B).

EXAMPLE 3

[0103] Cell lysates were labeled with an irreversible cysteine-reactive
iodoacetyl TMT reagent, digested, and then captured with the anti-TMT
resin. The bound peptides were eluted non-specifically with different pH
and detergent conditions or specifically with different concentrations
and pH of DMP elution reagent (FIG. 4A). Non-specific elution conditions
resulted in very poor elution efficiency of the alkylated peptides, and
the specificity of elution was less than 5%. In contrast, the DMP reagent
efficiently eluted over 100 times as many peptides with greater than 90%
specificity. Elution with 10 mM DMP was even more efficient with high pH,
but the high pH resulted in lower specificity of elution. As DMP is not a
biological molecule, there is no interference from endogenous levels. In
contrast, biotinylated molecules are more difficult to recover and may
result in the enrichment of interfering endogenously biotinylated
molecules.

EXAMPLE 4

[0104] The cysteine-reactive DMP tags and affinity enrichment reagents are
used for the enrichment, detection, and quantification of low abundance
protein modifications. A cell lysate was S-nitrosylated in vitro with
nitroglutathione, remaining free thiols were alkylated with
iodoacetamide, the nitroso groups were selectively released with ascorbic
acid, the newly exposed cysteines were alkylated with iodoTMT, and the
samples were then reduced, alkylated with iodoacetamide, and digested
with trypsin. These peptide samples were then enriched with the anti-TMT
antibody, and a sample of the load, the flowthrough, and the non-specific
and specific elutions were assessed for peptide recovery and specificity
(FIG. 4B). Even after in vitro labeling as a positive control, this
modification is undetectable without enrichment. The specific elution
with DMP yielded approximately 50% more peptides than a low pH elution,
and the specificity of the enrichment of iodoTMT-labeled peptides
increased from 6% to 21% with specific elution.

EXAMPLE 5

[0105] Multiplexed enrichment of low abundance protein modifications is
essential for the elucidation of mechanisms, pathways, and downstream
effects of biological signaling. Low abundance modifications are
difficult to detect and quantify, and multiplexed quantitative methods
that reduce variability introduced during sample handling are beneficial.
The combined use of isobaric versions of a cysteine-reactive DMP reagent
with multiplexed enrichment, efficient elution, and multiplexed
quantification allowed the analysis of replicates from multiple
experiment conditions, such as the enrichment of S-nitrosylated peptides
from in vivo and in vitro stimulated cells. S-nitrosylated peptides from
more than 80 proteins were identified and quantified across conditions,
and many of these peptides appeared to be regulated by both in vivo
treatment of cells with lipopolysaccharide and by in vitro
S-nitrosylation with nitrocysteine (FIG. 5).

EXAMPLE 6

[0106] Shorter linkers are beneficial for peptide capture and
identification, while longer linkers are beneficial for intact protein or
cell capture. Successful dentification of peptides by mass spectrometry
is affected by the molecular weight of covalently attached tags, such
that longer tags result in lower identification rates (Pichler, 2010). In
contrast, longer chain linkers connecting the reactive and epitope
regions of the tag may provide greater accessibility for buried sites.
For example, a chemical probe that covalently labels the catalytic active
site of an enzyme may have greater reaction rates and/or capture
efficiency for the intact protein when a longer polyethylene glycol
spacer separates the reactive and epitope ends of the probe because of
the improved accessibility of the reactive end to the reaction site
and/or greater accessibility of the epitope after labeling (Kidd 2001).

EXAMPLE 7

[0107] Labeling of oxidized proteins with hydrazide and alkoxy reagents,
digestion, capture, and quantitation to identify sites prone to oxidative
damage under multiple conditions. The ability to identify proteins and
protein sites prone to oxidative damage, such as carbonylation, is
important for the study of aging and degenerative disease states, and to
characterize biologically active therapeutics, including therapeutic
antibodies and bioactive protein-based hormones. For example, proteins
prone to oxidative damage may provide important insights into the
mechanisms of aging and disease, and multiple proteins have been
identified that are oxidatively damaged during aging (Feng 2008). The
ability to identify and quantify the specific sites of oxidative damage
with efficient and specific capture and recovery of modified and labeled
peptides provides detailed insights into the mechanisms of aging and
toxicity. The structural mapping of proteins and protein interactions can
be studied by mapping sites accessible to protein oxidation in the
presence of multiple partners and conditions including concentration
dependence and time-courses (Xu 2007). These sites of oxidation are
labeled with a set of affinity tags, the samples are mixed digested, and
labeled peptides are enriched and quantified to provide insights into
protein conformation and interactions.

EXAMPLE 8

[0108] Cell surface labeling with S-NHS or S-TFP versions of these
affinity tags are used for identification and quantification of cell
surface proteins that are differentially present on the surface in
response to treatment conditions, such as drug, toxin, hormone,
differentiating conditions, or environmental stressors (e.g. pH,
osmolarity, temperature, pO2). Cultured cells are treated with
control and one or more treatment conditions, including time-course and
dose-response, and then multiple isomers or analogs of the S-NHS or S-TFP
reagents are used to label cell surface proteins (FIG. 9). The samples
are then mixed before or after lysis and solubilization, and then labeled
proteins are enriched, reduced and alkylated to break disulfide bonds,
digested with a protease such as trypsin, and then quantitatively
analyzed by mass spectrometry to identify cell surface proteins that
change in response to treatment or differentiation.

EXAMPLE 9

[0109] Bioorthogonal labeling is a strategy that introduces non-native
chemical functionality into naturally occurring biomolecules of a living
system. This includes labeling with non-native derivatives of amino
acids, sugars, lipids, and precursor molecules with azide, alkyne, cyclic
alkyne, or phosphine functional groups (FIG. 8). In one example, cells
are grown in the presence of azido-sugars, including N-azido
acetylgalactosamine, N-azido acetylglucosamine, or N-azido
acetylmannosamine. These sugars are incorporated into the complex glycans
of glycosylated proteins. Cells treated with multiple conditions, such as
drug, toxin, hormone, differentiating conditions, or environmental
stressors, and then solubilized and then labeled via the azide-sugars
with a set of isomers or analogs of the phosphine or alkyne reagents.
These samples are combined and the glycosylated proteins are enriched in
one capture experiment with the affinity tag, antibody, and soft elution
competitor. These enriched proteins are then reduced, alkylated, and
digested prior to identification and quantification by mass spectrometry.

EXAMPLE 10

[0110] The disclosed gentle release reagents allow sensitive biomolecules,
including intact proteins and cells, to be enriched and purified without
damage from harsh elution conditions. An antibody specific for a cell
surface marker is labeled with an affinity tag, and then this antibody is
incubated with a suspended mixture of cells, such as from a blood sample.
An antibody specific for the affinity tag is immobilized on magnetic
particles, and these particles are incubated with the sample to enrich
cells expressing the specific antigen. After cell enrichment, the
immobilized antibody-affinity tag interaction is competitively displaced
with the DMP elution reagent(s) under otherwise native, non-destructive
conditions. The purified cells are then cultured and expanded for further
study.

EXAMPLE 11

[0111] Studies of transient or weak protein interactions often utilize
protein crosslinkers to stabilize the interactions, to provide molecular
detail of the regions or positions of interaction, and to provide
distance information by using crosslinkers of various lengths. These
crosslinked peptides are relatively rare because of the low labeling
efficiency and the complexity of the labeled samples, so various
strategies have been developed to improve the analysis. Isotopically
paired crosslinkers differing only in the number of heavy stable isotopes
in their chemical composition is a powerful identifier of low abundant
peptides, as singly crosslinked peptides will appear as distinct doublets
in the mass spectrometer (Muller 2001). Trifunctional crosslinkers with
affinity tags have also been utilized to allow enrichment of crosslinked
peptides from the complex background (Tang 2009). Isotopically labeled
trifunctional crosslinkers that consist of two reactive groups and the
affinity tag are presented in which the reactive groups are separated by
different chain lengths and one or more bonds that are more labile bonds,
such as disulfide or hydrazone bonds (FIG. 10). In this example,
isotopically labeled trifunctional crosslinkers with the soft elution tag
are used to crosslink proteins in multiple conditions (e.g. interaction
partner concentration, crosslinker concentration, time course, etc.). The
samples are pooled, reduced, alkylated, and digested, and then
crosslinked peptides from multiple treatments are simultaneously enriched
for analysis. The presence of selectively labile positions in the
crosslinker allows enriched, crosslinked peptides to be selectively
fragmented at these positions in a trap-based mass spectrometer capable
of MS3 fragmentation, and the resulting fragments can be re-isolated for
subsequent higher energy fragmentation for peptide sequence assignment
(Gardner 2010, Wu 2009). In this manner, the positions of chemical
crosslinking can be identified, optimized, and verified under multiple
conditions concurrently.

[0113] An isobaric, cysteine-reactive TMT reagent containing an
iodoacetyl-reactive group (iodoTMT®) was previously used for
irreversible labeling cysteine-containing peptides (FIG. 13). FIG. 13
shows chemical structures for iodoacetyl tandem mass tags (iodoTMT)
labeling in one embodiment. These tags are analogous to the cysTMT
reagents, except that the iodoacetyl chemistry is used to label thiols
irreversibly. Because cysteine-containing peptides in proteomic samples
are present in relatively low abundance, selective enrichment of labeled
peptides was required for quantification. When iodoTMT was used to label
different cysteine-modified peptide subpopulations, there was an even
greater challenge in sample enrichment selectivity.

[0114] For iodoTMT-labeled peptides, an anti-TMT antibody resin is used
for peptide capture with a novel TMT elution buffer using small molecule
analogs of the TMT reagent reporter region. The inventive reagent
provided selective, competitive elution of iodoTMT-labeled peptides (FIG.
14). FIG. 14 shows an example work flow for mass spectrometry analysis
using iodoTMT reagents. The iodoTMT reagents are less sensitive to
quenching by tris(2-carboxyethyl)phosphine (TCEP), so that the TCEP
reducing agent does not need to be removed by dialysis or desalting
before labeling with iodoTMT chemical tags.

[0116] The inventive iodoTMT reagent labeling was specific and efficient
for cysteine sulfhydryl residues with reactivity similar to iodoacetamide
(FIG. 15). FIGS. 15A-B show iodoTMT reagent labeling efficiency and
specificity. The iodoTMT reagent labeling is sensitive to stoichiometry
and pH conditions. Excess reagent or reaction conditions significantly
higher or lower than pH 8.0 will result in non-selective labeling at
lysines, histidines, aspartates, and glutamate residues. Because iodoTMT
reagent labeling was covalent and irreversible, reducing agents did not
need to be removed from protein samples before labeling. IodoTMT reagents
were also used for quantifying cysteine modifications such as
S-nitrosylation, oxidation, and di-sulfide bridges.

[0117] The enrichment workflow permitted cysteine-containing peptide
multiplex quantitation by LC-MS. Enrichment of iodoTMT-labeled peptides
was facilitated using an anti-TMT antibody raised against the reporter
region of the TMT reagents. Elution buffers with low pH or containing
denaturants (e.g. urea or SDS) that disrupt antibody-antigen interactions
are sometimes used for peptide elution; they also elute any non-specific,
unlabeled peptides bound to the resin.

[0118] To improve iodoTMT-labeled peptide elution specificity, various
small molecule analogs of the TMT reagent reporter region as potential
compounds for competitive elution were investigated. Using these analogs,
structural features of the anti-TMT antibody recognition site were
identified by SPR using a TMT-derivatized Biacore chip. The relative
affinity of each analog was also determined for the anti-TMT binding by
competitive elution. The inventive TMT elution buffer resulted in
increased labeled peptide enrichment specificity from 80% to 96% using
total cysteine-labeled peptide samples (FIG. 16). FIGS. 16A-B show
iodoTMT-labeled peptide enrichment and protein identification comparison.
While only a subset of peptides contain cysteine residues, the reduction
in complexity allowed by selective labeling and enrichment of
cysteine-containing peptides results in a greater number of proteins
identified from a given number of peptides identified.

[0119] IodoTMT reagents were also used as a probe for labeling
S-nitrosylated cysteines in a modified S-nitro switch assay. IodoTMT
reagents successfully labeled S-nitrosylated cysteines after selective
reduction using ascorbate, shown by an anti-TMT antibody Western blot
(FIG. 17). FIG. 17 shows an example of S-nitrosylation Western blotting.
A complex cell lysate was treated in vitro with vehicle (lanes 1,2) or
nitroglutathione (lanes 3,4) and then free thiols were blocked with
iodoacetamide. Nitrosylated thiols can then be labeled with iodoTMT after
the selective reduction of nitrosylated thiols with ascorbate (lanes
2,4). Enrichment specificity using low pH and TMT analog elution buffers
for S-nitrosylated samples also resulted; there was a 50% increase in
iodoTMT-labeled peptide identification by LC-MS using the inventive TMT
elution buffer (FIG. 18) which shows improved selectivity of enrichment
of S-nitrosylated peptides with a soft competitive elution strategy
instead of acidic elution buffer.

[0122] Similar to amine-reactive TMT reagents, iodoTMT reagents are a set
of six isobaric mass tags used to label different samples that are
combined for a relative quantitation of peptides in a single MS analysis.
Differential analysis of human serum by MS is challenging due to high
concentrations of albumin and IgG. Removal of these proteins using
various depletion strategies is often essential to study low-abundant
proteins. Serum sample preparation MS workflow consisted of
antibody-based Top 12 abundant protein depletion, iodoTMT reagent
labeling/enrichment, and LC-MS analysis.

[0124] A serum sample MS workflow combined top 12 abundant protein
depletion with iodoTMT reagent labeling and enrichment. This workflow is
similar to previously published workflows using ICAT reagents but enables
sixplex relative quantitation using LC-MS/MS. Three different
antibody-based depletion resins for serum sample preparation are
compared. The Pierce Top 12 Abundant Protein Depletion Spin Columns
provides equivalent depletion of abundant serum proteins but in a more
convenient single use format at a significant lower cost per sample.

[0125] Depleted serum samples are also labeled using iodoTMT reagents to
determine differences in relative protein abundance between normal and
disease samples. Analysis of iodoTMT-labeled samples before and after
enrichment with anti-TMT antibody resin show increased quantifiable
peptides, from <30% before enrichment to >90% after enrichment. The
high specificity of iodoTMT-labeled peptide enrichment is further
enhanced using a competitive anti-TMT antibody elution buffer. By
combining abundant serum protein depletion with iodoTMT reagent-based
quantitation, identification and quantitation of lower abundance serum
proteins are significantly improved.

EXAMPLE 14

[0126] Labeling of oxidized proteins with hydrazide and alkoxy reagents,
digestion, capture, and quantitation identified sites prone to oxidative
damage under multiple conditions. Protein oxidative damage results in
carbonylation and incorporation of reactive aldehyde and ketone groups
into amino acid side chains (Xu 2007). This oxidative damage can occur in
vivo in response to environmental stresses, resulting in permanent damage
to proteins. The ability to identify proteins and protein sites prone to
oxidative damage, such as carbonylation, is important for the study of
aging and degenerative disease states, and for characterizing
biologically active therapeutics including therapeutic antibodies and
bioactive protein-based hormones. For example, proteins prone to
oxidative damage may provide important insights into the mechanisms of
aging and disease, and multiple proteins have been identified that are
oxidatively damaged during aging (Feng 2008). Protein oxidation may be
induced in vitro accidentally during preparation or intentionally with
heavy metal-catalyzed production of free radicals, by ionizing radiation,
or by the addition of peroxides or other free radical producing chemicals
to modify proteins at accessible sites for protein structural mapping and
protein interaction studies. These modifications may be selectively
labeled with alkoxyamine or hydrazide reagents (FIG. 19). FIG. 19 shows
the chemical reactivity of hydrazide and alkoxyamine reagents for the
selective labeling, enrichment, identification, and quantification of
peptides, glycans, lipids, steroids, nucleotides, and other biomolecules
containing aldehydes and ketones. Sets of chemical tags containing
alkoxyamine or hydrazide groups can be used in a multiplexed fashion to
label samples produced from different treatment conditions or
time-courses (FIGS. 20, 21). These samples can be labeled under
denaturing conditions, mixed, processed, enriched, and analyzed as one
sample by LC-MS/MS (FIG. 22). FIG. 22 shows an example work flow for mass
spectrometry analysis of proteins using oxyTMT reagents. This workflow
allows the exact sites of modification to be identified and
simultaneously quantified across the experimental conditions to provide
insights into disease, sample preparation and manufacturing processes,
protein conformation, and interactions.

[0127] A method for profiling carbonylated peptides used a novel TMT
capable of determining sites of modification and multiplexed
quantification. Protein carbonylation is a common post-translational
modification that is linked to many diseases. Technologies for protein
carbonylation fall short because they do not allow multiplexed
quantitative comparisons, and are limited for determining site and type
of modifications. Isobaric Tandem Mass Tags (TMT) reagents (Thermo
Fisher) offer advantages to quantitative proteomics, distinctly
multiplexed quantification, and sequence identification. TMT consists of
a reactive group for covalent labeling of proteins or peptides, a
reporter group for relative quantification after peptide fragmentation
(mass between 126 and 131 Daltons), and a cleavable linker to keep the
overall mass constant (isobaric). A novel aldehyde-reactive tandem mass
tag, oxyTMT, was used for labeling based on an aminoxy functional group
using tandem mass spectrometry.

[0128] Carbonylated proteins were prepared using bovine serum albumin
(BSA) as standard protein and mouse liver mitochondria as complex lysate
reduced with TCEP; these were labeled with 4-hydroxynonenol (HNE) which
is widely used in the study of oxidative damage because it modifies
cysteines, lysines, and histidines. A liver mitochondria sample was not
labeled with HNE as an endogenous carbonylated sample. After assessing
labeling conditions, proteins were labeled with 10 mM TMT reagent in MES
buffer, pH 5. Labeled proteins were alkylated with iodoacetamide and
enzymatically digested with trypsin. Labeled peptides were passed through
anti-TMT resin, eluted with elution buffer and desalted by C18 Stage Tip.
Peptides were analyzed by LC-MS/MS using ESI LTQ Orbitrap Velos mass
spectrometry. All data were searched by ProteinPilot program (version 4.5
AB Sciex) against a mouse database containing BSA sequence.

[0129] Initial optimization was performed with analysis of standard,
single protein BSA, with carbonyl modifications introduced by HNE
modification by in vitro incubation with excess HNE, an
aldehyde-containing lipid metabolic compound, in physiological PBS
buffer. Carbonyl modification of BSA was confirmed by gel electrophoresis
and Western blot using anti-HNE antibody. Modified BSA was incubated with
excess TMT reagent, dialyzed, and digested with trypsin, and the TMT
labeled carbonylated peptides enriched using anti-TMT resin followed by
LC-MS/MS analysis by ESI LTQ Orbitrap Velos mass spectrometry. Expected
mass shifts on susceptible amino acids to HNE modifications, including
the oxyTMT label, were used to identify modified peptides by sequence
database searching using ProteinPilot program.

[0130] At least ten peptides labeled with oxyTMT were identified with 99%
probability in BSA-HNE-oxyTMT enriched sample. The unlabeled BSA
(control) showed no labeled peptides (data not shown). MS/MS spectra of
each labeled peptide was confirmed manually to have both oxyTMT reporter
ion (m/z=126) and expected modification mass shifts. Factors, such as
buffer pH, that might affect reactivity of the aminoxy with carbonyls
were assessed, and pH 5 was selected, as well as reaction conditions
using high amounts of SDS denaturant, to mimic conditions of protein
isolation for membrane proteins that are prominent in enriched
mitochondrial samples.

[0131] With optimized conditions for labeling and enrichment using the
oxyTMT reagent, labeling conditions were applied in mouse liver
mitochondria protein isolates labeled with HNE as exogenous sample and
without HNE as endogenous sample. Western blots were performed using
anti-TMT antibody as the primary antibody to visualize carbonylated
proteins in standard protein and complex mitochondrial lysate (data not
shown).

[0132] The data demonstrated oxyTMT as a novel solution for identification
and quantification of proteins susceptible to carbonylation, providing
new discoveries into the role of these modifications.

[0134] Applicants incorporate by reference the material contained in the
accompanying computer readable Sequence Listing identified as
Sequence_Listing_ST25.txt, having a file creation date of Mar. 8, 2013,
2:50 p.m., and a file size of 893 bytes.

[0135] The embodiments described in the specification are only specific
embodiments of the inventors who are skilled in the art and are not
limiting. Therefore, various changes, modifications, or alterations to
those embodiments may be made without departing from the spirit of the
invention or the scope of the following claims.